The folding of RNA into compact 3D structures is a hierarchical process in which the formation of RNA helices is followed by the formation of tertiary RNA motifs that specify the positioning of the helices within the structure. Due to the folding process of RNA, modular tertiary motifs have likely emerged for the purpose of adopting specific topological arrangements of helices. RNA motifs are defined by sequence signatures that correspond to a limited set of conserved and semi-conserved nucleotides specifying well-defined 3D conformers. Recent developments in RNA architectonics, an approach for rationally designing 3D RNA architectures, have established that RNA structure information can be implemented into an RNA sequence to direct its tertiary folding and supramolecular assembly with a high degree of control and predictability. Nevertheless, knowledge about the kinetics, thermodynamics and autonomous folding properties of most RNA tertiary motifs remains scarce, presently limiting their use as building blocks for nano-construction.
DNA has been extensively used as a medium for constructing nanoarchitectures. To build a DNA polyhedra two different design approaches have been used, which involves the use of single stranded DNA or identical tiles that are generated from single stranded DNA. Using the former strategy a DNA polyhedra with the connectivity of a cube, a truncated octahedron, a regular octahedron, a DNA cage in the shape of a tetrahedron and a bipyramid have been reported; however many of the DNA structures that have been reported suffer from poor assembly yields due to unspecific assembly of the building blocks, which increases the instability of the constructed nanoparticles.
While being more chemically labile than DNA, RNA molecules exhibit complex tertiary structures and provide a large repertoire of novel RNA-RNA interaction motifs that can be used as a medium, to construct a variety of highly complex architectures. Also, while RNA architectures are programmable like DNA, they can be more readily expressed in vivo. Moreover, natural RNA molecules display interesting functionalities that can be encoded within the RNA assemblies such as aptamers, or ribozymes. Compared to protein cages, nanocages made of RNA might induce a lower immune response, thus reducing the antibody production that leads to the clearance of the foreign nanoparticle. The organization of RNA duplexes in the shape of specific symmetrical 3D architectures is an alternative way of RNA packaging in living organisms. The 3.0 A resolution crystal structure of dodecahedral cage of duplex RNA, which is located inside the viral capsid of Pariacoto virus, is the only reported natural RNA polyhedral structure. However, this RNA cage is not thought to be stable in the absence of proteins. Previous studies have demonstrated that RNA can be designed as rigid modular units to construct filaments, and a variety of self-assembling programmable 2D arrays. Recently, the Φ29 packaging RNA complex was engineered to form functionalized 2D trimeric nanoparticles that deliver siRNA to induce apoptosis in cancer cells.
The rapidly expanding field of nanobiology opens up the possibilities for the development of new methods and compositions that can be used for the diagnosis, prognosis, and treatment of various diseases such as cancer. However, while an increasing number of novel drugs and therapeutic agents are being discovered, the problem of delivering them specifically to the desired site or cell has not been solved. RNA nanoparticles have been shown to be able to carry multiple components, including molecules for specific cell recognition, image detection, and therapeutic treatment. The use of such protein-free nanoparticles holds the promise for the repeated long-term treatment of chronic diseases with low immune response and should avoid the problems of short retention time of small molecules and the difficulty of delivery of particles larger than 100 nanometers.
Nanoparticles are ideal drug delivery devices due to their novel properties and functions and ability to operate at the same scale as biological entities. Nanoparticles, because of their small size, can penetrate through smaller capillaries and are taken up by cells, which allow efficient drug accumulation at the target sites (Panyam J et al., Fluorescence and electron microscopy probes for cellular and tissue uptake of poly (D, L-lactide-co-glycolide) nanoparticles, Int J Pharm. 262:1-11, 2003). There are several issues that are important for efficient design and drug delivery by nanoparticles, including the efficient attachment of drugs and vectors, controlled drug release, size, toxicity, biodegradability, and activity of the nanoparticle. Moreover, for successful design one needs to understand and control the intermolecular associations, based on natural favorability of interactions and various physical components.
Targeted delivery of nanoparticles can be achieved by either passive or active targeting. Active targeting of a therapeutic agent is achieved by conjugating the therapeutic agent or the carrier system to a tissue or cell-specific ligand (Lamprecht et al., Biodegradable nanoparticles for targeted drug delivery in treatment of inflammatory bowel disease, J Pharmacol Exp Ther. 299:775-81, 2002). Passive targeting is achieved by coupling the therapeutic agent to a macromolecule that passively reaches the target organ (Monsky W L et al., Augmentation of transvascular transport of macromolecules and nanoparticles in tumors using vascular endothelial growth factor, Cancer Res. 59:4129-35, 1999). Drugs encapsulated in nanoparticles or drugs coupled to macromolecules such as high molecular weight polymers passively target tumor tissue through the enhanced permeation and retention effect (Maeda H, The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting, Adv Enzyme Regul. 41:189-207, 2001; Sahoo S K et al., Pegylated zinc protoporphyrin: a water-soluble heme oxygenase inhibitor with tumor-targeting capacity, Bioconjugate Chem. 13:1031-8, 2002).
It would be desirable to possess multifunctional engineered nanoparticles and complexes that are capable of bypassing biological barriers and have low immune response to deliver multiple therapeutic agents into specific cells and tissues. Accordingly, a safe and efficient nanoparticle needs to be designed for the delivery of effective therapeutic and diagnostic RNAs.